Method for inducing sustained immune response

ABSTRACT

A method for promoting a sustained increased level of T-cell production in immunocompromised subjects in which method enkephalin peptides are administered according to an intermittent dose schedule. In particular, the method involves treatment of immunocompromised patients which includes the administration of enkephalin, either alone or in conjunction with other therapies, in an initial dosage regimen, with periodic booster dosages of enkephalin as necessary to maintain sustained immune system response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 10/146,999filed on May 16, 2002, which claims the benefit of priority of U.S.Provisional Application No. 60/291,237, filed on May 16, 2001. All ofthe above applications are hereby expressly incorporated by referenceinto the present application.

FIELD OF THE INVENTION

The present invention relates to methods of stimulating and promoting asustained natural immune system response, resulting in increasedresistance and inhibition of infectious agents, including viruses,bacteria, fungi and parasites, and other immunodeficiency-relatedailments. More specifically, the invention relates to an intermittentdose schedule for promoting a sustained increased level of T-cellproduction (cytotoxic T cells).

BACKGROUND OF THE INVENTION

The immune system protects the body against infectious agents, includingbacteria, viruses, fungi, and parasites. In addition, the immune systemprotects against cancer, as well as disease states that result fromimmune imbalance, opportunistic infections, and autoimmune disorders(Penney, U.S. Pat. No. 5,980,913). Stimulation of the immune system bypharmaceuticals is an important approach to the prevention and treatmentof agents that cause immune suppressed states.

The response by the immune system to an immunogen may be depressed as aconsequence of certain diseases or pathological conditions. For example,patients infected with the human immunodeficiency virus (HIV-1) maydevelop acquired immune deficiency syndrome (AIDS) or AIDS relatedcomplex (ARC), and thus have depressed immune responses. This patientclass is more susceptible to pathological infections or malignanciesagainst which a normal immune system would have otherwise providedsufficient protection. Other such immunocompromised individuals includepatients with cancer, or undergoing x-ray, surgery, or chemotherapytreatment.

Current treatments used to prevent the development of immunodeficiencyin individuals with viral infections, HIV for example, usually involveadministration of compounds that inhibit viral DNA synthesis therebyslowing onset of viral-related immunosuppression. Treatments forHIV-infected patients often involves administration of compounds suchas, for example, 3′-azido-3¹-deoxythymidine (AZT), 2′,3′-dideoxycytidine(DDC) and 2′,3′-dideoxyinosine (DDI), zidovudine, didanosine,zalcitabine, stavudine, and viramune. More recent treatments against HIVinclude administration of protease inhibitors such as, for example,saquinovir, nefinavir, ritonavir, indinavir, and others. Cytokinetherapy is also used in the treatment of AIDS patients, with researchgroups having demonstrated efficacy of interleukin-2 (IL2) in elevatingthe CD4 T-cell subset in HIV positive patients (Kovacs, et al., N. Engl.J. Med., 1996; 335: 1350-1356). Reports have detailed that IL2 can alsoincrease CD8 T-cell count (Schmitz, et al., Science, 1999; 283:857-860). Unfortunately, the use of IL2 is normally accompanied by majortoxicity (Davey, et al., JAMA, 2000; 284: 183-189). Nevertheless, giventhe potential promise of these therapies directed toward anti-retroviraleffects, none have proven to be totally effective in treating orpreventing development of AIDS. In addition, many of these compoundscause adverse side effects including low platelet count, diarrhea,nausea, renal toxicity, and bone marrow cytopenia (Kempf, et al., U.S.Pat. No. 6,017,928; Lai, et al., U.S. Pat. No. 6,093,743). Numerousclinical studies with methionine enkephalin (met-enkephalin) in normalvolunteers, HIV positive, and cancer patients showed no major toxicity(Plotnikoff, et al, Clin. Immun. Immunopath., 1997). However, themeasured half-life of met-enkephalin in plasma is approximately 2minutes (Bihari, et al., Seventh Int. Conf. On AIDS, 1991). Thus, thereexists a need in the art for improved methods of stimulating a sustainedimmune system response in patients in need of such treatment, such aspatients include those with compromised immune system responses (e.g.AIDS), or the potential to develop compromised immune system responses(e.g. HIV-infected patients).

SUMMARY OF THE INVENTION

All cited patents, patent applications and references are herebyincorporated by reference in their entirety.

Recent studies of met-enkephalin indicated that met-enkephalin activatedgene transcription of IL2 (Wybran, et al., from Some ImmunologicalEffects of Methionine-Enkephalin In Man: Potential Therapeutical UseLeukocytes and Host Defense. 205-212, Alan R. Liss, Inc. 1986) and gammainterferon (Brown, et al., Immunology, 1986; 103:19-26) and IL 12,(Zhong, et al., Augmentation of TNF-alpha Production, NK cell activityand IL-12 p35 mRNA Expression by Metliaonine Enkephalin, 1996; 17(2):182-5) AIDS patients have a deficiency of IL2, and gamma interferon(Fauci, et al., Science, 1993; 262: 1011-1018). Recently HIV positiveshave shown a deficiency of met-enkephalin (Valentine, et al., FASEB J.,1988; 2(5): 4518; Chao, Thesis, University of Illinois College ofPharmacy 1993). All of the above cytokines are derived from prohormonesin T helper cells (Plotnikoff, et al., Clin. Immun. Immunopath., 1997;82(2): 93-101). Immune suppression is, in part, a consequence ofcyctokine deficiency (Fauci, et al., Science, 1993; 262: 1011-1018).

Based on the above, however, there would be no expectation by one ofskill in the art that the active agents of the present invention couldbe used in methods of treatment useful in producing a sustained immuneresponse in a patient comprising administering the active agents on anintermittent dosage schedule to a patient in need of such treatment.

The present invention is based on the surprising discovery that aregular dosing schedule of met-enkephalin is effective in promoting asustained cell increase in immune system response including sustainedcell levels, in a patient for at least one month after cessation of thedosing.

The present invention provides, inter alia, for methods of treatmentuseful for inducing a sustained immune system response in animmunocompromised patient in need of such treatment wherein the methodcomprises administering to the patient an effective amount of anenkephalin peptide, either alone, combined, or in further combinationwith other compounds useful for increasing immune system response,including vaccines. In this context, “immunocompromised” refers to anyreduction in T-cell number or function.

The present invention also provides, inter alia, for methods oftreatment useful for inducing a sustained immune system response in anHIV-infected patient, wherein the method comprises administering to theHIV-infected patient an effective amount of an enkephalin peptide,either alone, combined, or in further combination with other compoundsuseful for slowing the progression of HIV proliferation orHIV-associated infections, including reverse transcriptase inhibitorssuch as 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxycytidine (DDC)and 2′,3′-dideoxyinosine (DDI), zidovudine, didanosine, zalcitabine,stavudine, and viramune; protease inhibitors such as saquinovir,nefinavir, ritonavir, and indinavir; cytokines such as G-CSF, IL-11,IL-12, IL-2; and gamma interferon and antibiotics or other drugs usedfor the treatment or prevention of infections in HIV-infected patients.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

Unless otherwise indicated, the term “active agents” as used hereinrefers to the group of compounds comprising the class of enkephalinpeptides.

Unless otherwise indicated, the term “enkephalin” or “enkephalinpeptides” includes any compound that falls into the general category ofopioid peptide molecules, including compounds having the followingpeptide structure at one terminus:

(SEQ ID NO: 1) Tyr-Gly-Gly-Phe-Rwhere R is either Met or Leu. Exemplary enkephalin peptides are shown inTable 1.

TABLE 1  Opioid peptides and their Precursors and structures PrecursorsPeptides Structures Pro- α-EndorphinTyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr- OpiomelanacortinPro-Leu-Val-Thr (SEQ ID NO: 2) (PCMC) γ-EndorphinTyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu (SEQ ID NO: 3) β-EndorphinTyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr- (human)Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu (SEQ ID NO: 4) Proenkaphalin A Leu-enkaphainTyr-Gly-Gly-Phe-Leu (SEQ ID NO: 5) Met-enkephalinTyr-Gly-Gly-Phe-Met (SEQ ID NO: 6) HeptapeptideTyr-Gly-Gly-Phe-Met-Arg-Gly (SEQ ID NO: 7) OctapeptideTyr-Gly-Gly-Phe-Met-Arg-Gly-Leu (SEQ ID NO: 8) Peptide ETyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu- (bovine)Trp-Trp-Met-Asp-Tyr-Gln-Lys-Arg-Tyr-Gly-Gly-Phe- Leu (SEQ ID NO: 9)Prodynorphin Dynorphin A(1-8)Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile (SEQ ID NO: 10) (Proenkaphalin B)Dynorphin A(1-17) Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-(porcine) Lys-Trp-Asp-Asn-Gln (SEQ ID NO: 11) Dynorphin B(1-13)Tyr-Gly-Gly-Phe-Leu-Arg-Gln-Phe-Lys-Val (porcine)Val-Thr (SEQ ID NO: 12) α-Neo-endorphinTyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys (SEQ ID NO: 13) β-Neo-endorphinTyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro (SEQ ID NO: 14) New dynorphinTyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val- (ieumorphin)Val-Thr-Arg-Ser-Gln-Glu-Asp-Pro-Asn-Ala-Tyr-Tyr- (porcine)Glu-Glu-Leu-Phe-Asp-Val (SEQ ID NO: 15) Others _(B)-CasomorphinTyr-Pro-Phe-Pro-Gly-Pro-Ile (SEQ ID NO: 16) DermorphinTyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂ (SEQ ID NO: 17) KyotorphinTyr-Arg (SEQ ID NO: 18)

Enkephalin analogues also fall within the scope of the invention, andare described in the following references which are incorporated byreference: U.S. Pat. No. 4,468,383, to Rodbard et al., issued Aug. 28,1984; U.S. Pat. No. 4,371,463, to Pert et al., issued Feb. 1, 1983; U.S.Pat. No. 4,261,883, to Smolarsky, issued Apr. 14, 1981; U.S. Pat. No.4,254,106, to Wilkinson, issued Mar. 3, 1981; U.S. Pat. No. 4,213,968,to Kastin et al., issued Jul. 22, 1980; U.S. Pat. No. 4,198,398, toHudson et al., issued Apr. 15, 1980; U.S. Pat. No. 4,127,534, to Coy etal., issued Nov. 28, 1978; U.S. Pat. No. 4,092,304, to Jones, Jr. etal., issued May 30, 1978; U.S. Pat. No. 4,028,319, to Jones, Jr. et al.,issued Jun. 7, 1977; J. Chang et al., “Opiate Receptor Affinities andBehavioral Effects of Enkephalin: Structure Activity Relationship of TenSynthetic Peptide Analogues,” 18 Life Sci. 1473-1482 (1976); G. A. Gacelet al., “Synthesis, Biochemical and Pharmacological Properties of BUBUC,a Highly Selective and Systematically Active Agonist for In Vivo Studiesof Delta-Opioid Receptors”, 11 Peptides 983-988 (1990); and B. P.Rogues, “Peptidomimetics as Receptor Agonists or Peptidase Inhibitors: AStructural Approach in the Field of Enkephalins, ANP and CCK”, 32Biopolymers 407-410 (1992).

As used herein, the term “sustained immune system response” is taken tomean maintaining in a patient an increase from the baseline of serumlevels of cells and molecules associated with the immune system,including T-cells such as CD3, CD4, CD8, CD56, CD 25, and CD38 andmolecules such as the interleukins and interferons.

As used herein, the term “baseline” or “base level response” is taken tomean the serum levels in a patient before administration of active agentof cells and molecules associated with the immune system, includingT-cells such as CD3, CD4, CD8, CD56, CD 25, and CD38 and molecules suchas the interleukins and interferons.

The phrase “intermittent dose schedule” of enkephalin peptides as usedherein refers to an initial routine of repeated administration of aenkephalin peptides, ranging from a daily to a weekly basis for somedefined period of time (or, alternatively, a compound that promotes invivo production of enkephalin peptides for some defined period of time)(collectively referred to as an “initial dosage regimen”), followed by aperiod of time when such administration is discontinued. Additionalenkephalin peptides (or compounds that promote in vivo production ofenkephalin peptides) are administered thereafter on an intermittentbasis.

Exemplary intermittent dose schedules include, but are not limited to,administration of enkephalin peptides from one to five times a week overthe course of a 12 week period, then discontinued for a period of timeranging from 4 to 24 weeks. Thereafter, based on some defined criteria,booster doses are given, up to 5 times a week for 1 to 4 weeks. Otherexamples of intermittent dose schedules that fall within the scope ofthis invention include administration of enkephalin peptides from 1 to 5times a week over 4 weeks, then discontinued for a period of timeranging from 4 to 16 weeks. Thereafter, booster doses are given, up to 5times a week for 1 to 4 weeks, depending on the level of sustainedresponse measured. Other intermittent schedules may also be utilized.

As used herein, the term “HIV” includes all variants and types of HIV-1,HIV-2, and other synonymous retroviruses, such as human T-lymphotropicvirus type III (HTLV-III) and lymphadenopathy associated virus (LAV-1and LAV-2).

As used herein, the term “AIDS” refers to acquired immune deficiencysyndrome, AIDS-related complex (ARC), and decreased lymphocyte numbersin HIV-infected individuals.

As used herein, the term “treating or preventing AIDS” includespreventing or decreasing the immunosuppression caused by AIDS, forexample, by decreasing HIV levels in the patient's peripheral bloodlymphocytes, or by increasing lymphocyte numbers; replenishing the bonemarrow; increasing survival of HIV-infected patients; as well aspreventing or decreasing the associated symptoms, disorders, andinfections associated with HIV infection, including but not limited tosusceptibility to pathogenic and opportunistic organisms and infections,anemia, thrombocytopenia, and lymphopenia.

As used herein, the term “opportunistic infection” refers to infectionswith an organism that would not normally be pathologic in patients withproperly functioning immune systems.

Many of the peptides contemplated in the instant invention arecommercially available, but alternatively may be synthesized by anyconventional method, including, but not limited to, those set forth inJ. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed.,Pierce Chemical Co., Rockford, Ill. (1984) and J. Meienhofer, HormonalProteins and Peptides, Vol. 2, Academic Press, New York, (1973) forsolid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol.1, Academic Press, New York, (1965) for solution synthesis. Thedisclosures of the foregoing treatises are incorporated by referenceherein.

In general, these methods involve the sequential addition of protectedamino acids to a growing peptide chain (U.S. Pat. No. 5,693,616, hereinincorporated by reference in its entirety). Normally, either the aminoor carboxyl group of the first amino acid and any reactive side chaingroup are protected. This protected amino acid is then either attachedto an inert solid support, or utilized in solution, and the next aminoacid in the sequence, also suitably protected, is added under conditionsamenable to formation of the amide linkage. After all the desired aminoacids have been linked in the proper sequence, protecting groups and anysolid support are removed to afford the crude polypeptide. Thepolypeptide is desalted and purified, preferably chromatographically, toyield the final product.

The peptides can synthesized according to standard solid-phasemethodologies, such as may be performed on an Applied Biosystems Model430A peptide synthesizer (Applied Biosystems, Foster City, Calif.),according to manufacturer's instructions. Other methods of synthesizingpeptides or peptidomimetics, either by solid phase methodologies or inliquid phase, are well known to those skilled in the art.

Alternatively, the peptides may be produced via conventional molecularbiological methods.

DESCRIPTION

In one embodiment of the invention an effective amount of active agentis administered to a patient once a week over the course of a 12 weekperiod, then stopped. Measurement of the patient's immune response(T-cell counts) is made from 4 to 16 weeks after cessation of theinitial dosing, and compared with both the base level response andresponse levels measured at the end of the dosing schedule. Thereafter,booster doses are given, as needed, up to 5 times a week for 1 to 4weeks.

In other embodiments of the invention an effective amount of activeagent is administered to a patient from 1 to 5 times a week over thecourse of a 4 week period, then stopped. Measurement of the patients'immune response (T-cell counts) is made from 4 to 16 weeks aftercessation of the initial dosing. Thereafter, booster doses are given, asneeded, up to 5 times a week for 1 to 4 weeks, depending on the level ofsustained response measured. Other intermittent schedules may also beutilized.

In one aspect of the present invention methods for treating orpreventing AIDS in an HIV-infected patient, comprising administering toan HIV-infected patient an amount effective to treat or prevent AIDS ofat least one compound selected from the group of active agents, alone,in combination with each other, or in combination with other compoundsthat are beneficial for treating or preventing AIDS in HIV-infectedindividuals, including but not limited to reverse transcriptaseinhibitors including but not limited to 3′-azido-3′-deoxythymidine(AZT), 2′,3′-dideoxycytidine (DDC) and 2′,3′-dideoxyinosine (DDI),zidovudine, didanosine, zalcitabine, stavudine, and viramune; proteaseinhibitors such as saquinovir, nefinavir, ritonavir, and indinavir;cytokines such as G-CSF, IL-11, IL-12 and IL-2 and erythropoietin; andantibiotics or other drugs used for the treatment or prevention ofinfections in HIV-infected patients or vaccines.

For use in treating or preventing onset of AIDS in an HIV-infectedindividual, the active agents may be administered by any suitable route,but are preferably administered either orally, parentally, by inhalationspray, transdermally, intravenously, rectally, intra-arterially,nasally, eye-drops, buccal patch or topically in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles. The term parenteral as used hereinincludes subcutaneous, intramuscular, intravenously, intra-arterially,or intratendinous.

The active agent may also be administered directly to the individual ina pharmaceutically suitable vehicle, for example, a solution of 5% DMSOor 10% ethanol in saline. In a preferred embodiment, multipleadministrations of the active agents are made over the period of timeencompassing effective treatment.

A large variety of alternatives are known in the art as suitable forpurposes of sustained release and are contemplated as within the scopeof the present invention. Suitable delivery vehicles include, but arenot limited to, the following: microcapsules or microspheres; liposomesand other lipid-based release systems; crystalloid and viscousinstillates; absorbable and/or biodegradable mechanical barriers; andpolymeric delivery materials, such as polyethylene oxide/polypropyleneoxide block copolymers (e.g. poloxamers), poly-orthoesters, cross-linkedpolyvinyl alcohol, polyanhydrides, polymethacrylate andpolymethacryladmide hydrogels, anionic carbohydrate polymers,polyethylene glycol, etc. Useful delivery systems are well known in theart and are described in, e.g., U.S. Pat. No. 4,937,254, the entiredisclosure of which is hereby incorporated by reference.

The active agents may be made up in a liquid form (e.g., solutions,suspensions, or emulsions), and may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional pharmaceutically acceptable adjuvants, such as stabilizers,wetting agents, emulsifiers, preservatives, cosolvents, suspendingagents, viscosity enhancing agents, ionic strength and osmolalityadjustors and other excipients in addition to buffering agents. Suitablewater-soluble preservatives which may be employed in the drug deliveryvehicle include sodium bisulfite, sodium thiosulfate, ascorbate,benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate,parabens, benzyl alcohol, phenylethanol or antioxidants such as VitaminE and tocopherol and chelators such as EDTA and EGTA. These agents maybe present, generally, in amounts of about 0.001% to about 5% by weightand, preferably, in the amount of about 0.01 to about 2% by weight.

For administration, the active agent is ordinarily combined with one ormore pharmaceutically acceptable adjuvants appropriate for the indicatedroute of administration. The compounds may be admixed with lactose,sucrose, starch powder, cellulose esters of alkanoic acids, stearicacid, talc, magnesium stearate, magnesium oxide, sodium and calciumsalts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, thecompounds of this invention may be dissolved in saline, water,polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidalsolutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil,tragacanth gum, and/or various buffers including phosphate bufferedsaline. Other adjuvants and modes of administration are well known inthe pharmaceutical art. The carrier or diluent may include time delaymaterial, such as glyceryl monostearate or glyceryl distearate alone orwith a wax, or other materials well known in the art.

The dosage regimen of the active agents for inducing sustained immunesystem response in HIV-infected patients is based on a variety offactors, including the age, weight, sex, medical condition of theindividual, the severity of the condition, the route of administration,and the particular active agent to be administered. Thus, the dosageregimen may vary widely, but can be determined routinely by a physicianusing standard methods. Dosage levels of the order of between 10⁻¹⁴μg/Kg to 30,000 μg/Kg of the active agents per body weight may be usedfor all methods of use disclosed herein. Preferred dosage levels rangefrom 1 μg/Kg to 250 μg/Kg in the intermittent dose schedules. In apreferred embodiment, dosages during the initial dosage regimen are 20μg/Kg bodyweight of the subject and intermittent dosages after suchinitial dosage regimen range from 20 to 100 μg/Kg bodyweight.

The efficacy of the dosing schedules are determined by methods thatmeasure indications such as decreases in HIV levels in the patient'speripheral blood lymphocytes, (viral load), anemia, thrombocytopenia,and lymphopenia; and increased CD4+, CD8+, CD3+, and CD56+ cell counts,lymphocyte numbers, antibody titer, resistance to pathogenic andopportunistic infections, and survival of HIV-infected patients.

The active agents of the present invention may also be administered in afurther stabilized form, such as, for example, associated withpolyethylene glycol or as a fusion protein, or other forms known in theart.

AIDS, surgery patients and cancer patients have been shown to haveelevated cortisol levels resulting in suppression of cytokineproduction, such as IL2 and gamma interferon. Such suppression ofcytokine production contributed to the suppression of the immune system.Cortisol, in particular, is believed to have inhibitory effect on genetranscription of IL2, and down-regulates cytotoxic cells and NKlymphocytes. See, e.g., K. Ogawa et al., “Suppression of CellularImmunity by Surgical Stress,” Surgery, 127(3): 329-36 (March, 2000);“Suppressant Effects of Cortisol,” Goodman and Gilmans, ThePharmacological Basis of Therapeutics, Pergamon Press, New York (8^(th)Ed. 1990). Cortisol also produces fatigue and weakness in AIDS patients.The studies reported in the examples demonstrate that an intermittentdosage regimen of met-enkephalin reverses HIV-induced immune suppressionresulting in increased IL2 levels and T cells and would be expected toreverse such immune suppression induced by a common pathway—cortisol—asseen in AIDS, surgery, cancer, and other patients.

The present invention may be better understood with reference to theaccompanying examples that are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention.

Example 1 In Vivo Administration of Met-Enkephalin as a ‘TherapeuticVaccine’

An original 12 week double blind study designed to measure the effectsof a regular dosing schedule of met-enkephalin on cytotoxic T cellslevels in HIV-infected patients was undertaken. Dosages for each patientvaried according to group: (1) 60 μg/Kg; (2) 125 μg/Kg and (3) placebo.The patients were administered either active agent (met-enkephalin) orplacebo (normal saline; control group) once a week for twelve weeks bymeans of intravenous infusions. At the eight and twelve week mark,samples from each patient were taken in order to measure levels ofT-cells. At twelve weeks the infusions were stopped. Measurement ofT-cell counts were again recorded 4 weeks after stopping the infusions(16 week time point). The results show sustained response levels ofcytotoxic T cells a month after the last dosing of met-enkephalin.

TABLE II Immunological Values One Month After Last N-Saline InfusionBASELINE ONE MONTH DIFFERENCE CD3 (6/8) 1260 ± 136 (1) 976 ± 103* −284CD4 (6/8)  383 ± 37 304 ± 37* −79 CD8 (5/8)  914 ± 153 668 ± 104* −246CD56 (7/8)  22 ± 6  80 ± 18* +58 CD25 (5/8)  45 ± 8  90 ± 17* +45 (IL2Receptor) CD38 (7/8)  220 ± 8 505 ± 84 +285 CD3 *P = 0.01 CD4 *P = 0.001CD8 *P = 0.02 CD56 *P = 0.01 CD25 *P = 0.02 CD38 *P = 0.01 (1) Mean cellnumbers is. e. per cu.mm. (N/8) Number of patients with cell countsabove (or below) baseline

TABLE III IMMUNOLOGICAL VALUES ONE MONTH AFTER LAST N-METHIONINEENKEPHALIN INFUSION BASELINE ONE MONTH DIFFERENCE CD3 (9/12) 1003 ±81(1) 1183 ± 116* +180 CD4 (7/12)  314 ± 39  351 ± 37* +37 CD8 (9/12) 659 ± 64  787 ± 79* +128 CD56 (11/12)  22 ± 6  137 ± 6* +115 CD25(10/12)  58 ± 12  145 ± 62* +87 (IL-2 receptor) CD38 (9/12)  320 ± 79 636 ± 103* +316 (1) Mean cell numbers of cells ± s.e. per cu.mm CD3 *P= 0.04 CD4 *P = 0.01 CD8 *P = 0.001 CD56 *P = 0.001 CD25 *P = 0.09 CD38*P = 0.05 (N/12) Number of patients with cell counts above baseline

Summary of Results:

Cytotoxic T cells (including CD3, CD4 and CD8 cells) were found to beelevated compared to baseline values one month after the last dosing ofmethionine enkephalin (8-12 weeks infusion). In sharp contrast theplacebo treated control group had a progressive decrease of the samecells over the 16 week study. The results indicate that treatment withmet-enkephalin increases cytotoxic T cells (that can reduce viral load)and may be considered as a “therapeutic vaccine” approach to thetreatment of HIV on an intermittent dosage schedule. These sustainedresults are particularly surprising given the measured half-life ofMet-enkelphalin in plasma is approximately two minutes.

CD3, CD4, and CD8 Cells

Patients treated with methionine enkephalin one month earlier were foundto have sustained increases of T cells compared to baseline (meanCD3+180, CD4+37, CD8+128). In sharp contrast patients infused withn-saline one month earlier were found to have sharp declines in T cellnumbers compared to baseline values (CD3−284, CD4−79−246).

CD56 Cells

Natural killer cells numbers were greater than baseline for both themet-enkephalin and n-saline infusion groups compared to baseline.However, the met-enkephalin group was significantly higher than then-saline group (80+18 versus 137+21 p=0.03).

CD25 Cells 1L2 Receptor

Interleukin II receptor expression was increased in both groups comparedto baseline although the met-enkephalin had a larger increase than then-saline group (45 versus 87 cells).

CD38 Cells

Both groups were found to have increases of CD 38 cell numbers abovebaseline (285 for the saline group versus 316 for the enkephalin group).

This study supports the hypothesis proposed by Walker and coworkers,that the most important cells in maintaining the immune system in AIDSpatients are the cytotoxic T cells (CD3, CD8) (Walker, et al., Nature,1987; 328: 345-348). These cytotoxic T cells are the same cellsactivated by vaccines (Belyakov, et al., J. Clin. Invest., 1998;102(112): 2072-2081). CD4 subsets also have cytotoxic actions againstHIV (Hahn, et al., Int. Rev. Immunol., 1999; 18(5-6): 449-464). NK cellsalso are cytotoxic against HIV (Melder, et al., FASEB J., 1989; 3: 4).

In the present study CD56 cells were elevated in both enkephalin andsaline treated groups (8-12 weeks of infusions). The significantlygreater increase in the enkephalin group can be attributed to theactivating effects of the enkephalin and resultant increases in theenkephalin group can be attributed on the NK cells. Saline infusions for8-12 weeks may also activate the same systems to some extent due to thestress of the procedure resulting in some increase of the cytokines andCD25.

Example 2 In Vivo Administration of Met-Enkephalin of Advanced AIDSPatients

Six advanced AIDS patients with CD4 Counts of less than 200 cells per μlwere treated with an intermittent therapy of met-enkephalin. During aninitial dosage regimen, dosages of met-enkephalin were given at 20 μg/Kgbodyweight of the subject three times per week for 4 weeks. Thereafter,subjects were given 20-100 μg/Kg bodyweight on an as-needed basis.Patients 4 and 6 had intermittent treatments after an initial dosageregimen.

Results at the start of the regimen, after one month of regular initialdosage regimen, and, for patients 4 and 6, after 3 additional months ofintermittent therapy are given in Table IV and V.

TABLE IV AIDS Patients Rx Regimen and Clinical Evaluation Met-EnkPatient Rx Treatment Associated Clinical Description Pre-Rx statusRegimen Duration Treatment Evaluation 1. 42 Yr. Male Kapos Sarcoma 20μg/Kg,  4 mos. Kaposi Sarcoma stable Homosexual (Cutancous) 3x/wk Noopportunistic infection Pheumocystis carinii (5/15/87) AIDS-(IV, C, D)AZT (5/87) 2. 42 Yr. Male Pheumocystis Carinii, 20 μg/Kg 10 mos.Amphotericin No opportunistic infection except African Esophageal 3x wk(12/85); Sulfadizine cardida esophagitis Candidiasis; Cerebral to 100μg/Kg Vincristine Shigella enteritis cured Toxoplastrosis; 3x/wkMiconazol Kaposi sarcoma stable Generalized is (10/87) Klebgiellasepticemia cured Cryptococcus (lung, G.I Bone Marrow Meningitis,Parlcarditis) Kaposi Sarcoma stable AIDS-(IV, C, D) 3. 34 Yr. MaleKaposi Sarcoma 20 μg/Kg, 24 mos. Local Liquid Kaposi sarcoma stable(some Homosexual (cutaneous lymph 3x wk (10/85); N2 Temission, some newlesions) nodes) to 100 μg/Kg; No opportunistic infections AIDS-(IV, C,D) 3x (10/87) Sulmonelia septicemia at month 22 4. 37 Yr. Cerebral 20μg/Kg, 3x  8 mos. Sulfidiazine Month 7 of RX Paraplegia and FemaleToxoplasmosis; wk/(1/87) to 100 μg/Kg Pyrimethamine Sphimeteral problems(HIV African Candida Esophagitis (2/87) radicultais?) (cutaneous) Deathof unknown origin (Bacterial Kaposi Sarcoma infection7 Lung emboli7)AIDS (IV, C) No recurrence of opportunistic infection 5. 34 yr-MaleCerebral 20 μg Kg), 3x/wk  4 mos. Sulfadiazine Signs of cerebral atrophyafter Homosexual Toxoplasntosis (1/07) to 50 μ/Kg (dropped Pyrimethamine2 mos. of RX (HIV dementia) AIDS (IV, C) 3x/1 wk(3/87) out) Norecurrence of opportunistic infection 6. 31 Yr. Non Hodgkin 20 μg/Kg, 11mos. Disappearance of skin lesions at first Male Lymphoma 3x/wk (4/86)(dropped (biopsy) but relapse (7 months) with Homosexual Cerebral to 100μg/Kg out) probable lung involvement at Toxoplasmosis (5/87) month 11Mycobacicrium No recurrence of opportunistic Kansami infection. KaposiSarcoma (Colandoris) AIDS-(IV, C, D)

TABLE V AIDS Patients Immunology Other Readings Other Readings Patient #Start 1 Month 4 Months (Months) (Months) Lymphocytes/mm³ 1 578 — 1130* 2700 1066* 1172* 540 (8 M)  360 (10 M) 3 1716 2080* 1776* 1173 (24 M) 41380 1440* 1620* 969 (7 M) 5 896  928*  360 — 6 247  324*  252 112 (11M) CD3/mm³ 1 387 —  881* — 2 616  820 1337 410 (8 M)  256 (10 M) 3 12871560* 1598* 961 (24 M) 4 730  940*  790* 532 (7 M) 5 689  677 — — 6 205 250*  164 78 (11 M) CD4/mm³ 1 29  68* — — 2 28  43*  49* 32 (8 M)   7(10 M) 3 205  437*  391* 106 (24 M) 4 40  40  60* 58* (7 M) 5 63  65* 50 — 6 25  32*  13 10 (11 M) Start 1 M 4 M CD8/mm³ 1 61  66* 2 546 767* 1271* 340 (8 M)   23 (10 M) 3 1047 1144* 1154* 821 (24 M) 4 650 860*  770* 610 (7 M) 5 609  594  198 — 6 205  250*  164 78 (11 M) RatioCD4/CD8 1 0.5 —   1.0* — 2 0.05   0.06*   0.40 0.09* (8 M) 0.02 (10 M) 30.02   0.38*   0.34* 0.13 (24 M) 4 0.06   0.05   0.08* 0.09* (7 M) 50.10   0.11*   0.26* — 6 0.10   0.13*   0.08 0.13* (11 M) PHA (cpm ×10³) 1 213 — — 2 42   8  28 15 (8 M)   11 (10 M) 3 144  72  128 227* (24M) 4 92  151*  187* 16 (7 M) 5 48  82*  60* — 6 96  86  37 8 (11 M)Start 1 Month 4 Months PWM (cpm × 10³) 1 32 — — — 2 14   6   7 12 (8)  9 (10 M) 3 14  19*  12 57* (24 M) 4 21  25*  47* — (7 M) 5 13  18*  12— 6 17  19* — — NK (%) 1 3 —  17* 2 27  27   6 — 3 26  43*  33* 13 (8 M)4 5   5   4 30* (24 M) 5 2   7* — 16* (7 M) 6 18  19* — — (11 M) IL2(Units) 1 0.04 —   0.1* — 2 0 — — 0.1* (8 M)   0 (10 M) 3 0.34   0.43*  0 — 4 0   0   0 0.03* (7 M) 5 0   0   0.03* — 6 0.1   0 — — (11 M)*Increase over baseline

Summary of Results:

The study described in this Example 2 demonstrates that intermittentdosage schedules provided comparable or better results in some patientsto patients with continued regimens comparable to the initial dosageregimen. In particular, patients 4 and 6 showed positive results over anextended time period, with no recurrence of opportunistic infection.Patient 6 had a disappearance of skin lesions for an extended period oftime. Changes included a visible reduction in size in some tumors, aswell as tumor color resolution to white.

Patient 4 demonstrated increased lymphocyte, CD3, CD4, and CD8 counts;Patient 6 experienced increased lymphocyte counts compared to baseline.The principal immunological effects were increases in T cell subsets(CD3, CD4 and CD8) and blastogenesis with activation of genetranscription.

Thus, this clinical data demonstrates that an intermittent dose therapyof met-enkephalin can increase gene transcription of T cells in advancedAIDS patients and reduce or stabilize Kaposis sarcoma nodules.

It will be recognized by those of skill in the art that the activeagents and methods of the present invention may be further modifiedwithout departing from the spirit and scope of the invention, and arenot limited by the foregoing examples or preferred embodiments. Thisdisclosure is intended to cover all variations, uses, or adaptations ofthe invention that generally follow the principles of the invention inthe art to which it pertains.

I claim:
 1. A method of treating cortisol-induced immunosuppression in asubject by: treating said subject with an initial dosage regimen ofenkephalin peptides; discontinuing said initial dosage regimen with nofurther administration of said enkephalin peptides for at least fourweeks; and administering booster dosages of enkephalin peptidesthereafter as necessary to reduce said cortisol-inducedimmunosuppression.
 2. The method of claim 1, wherein said initial dosageregimen comprises SEQ ID NO: 1, either alone or in conjunction withother therapies, wherein each dose in the initial dosage regimen is 1 to250 μg/Kg bodyweight of the subject, and wherein said initial dosageregimen reduces said cortisol-induced immunosuppression, wherein said atleast four weeks of discontinuing said initial dosage regimen with nofurther administration of said enkephalin peptide is for a period of 4to 24 weeks, wherein the reduction in said cortisol-inducedimmunosuppression is sustained during said period in which saidadministration of said enkephalin peptide is discontinued, and whereinsaid reduction in said cortisol-induced immunosuppression comprises anelevated amount of at least one selected from the group consisting ofIL2, gamma interferon, cytotoxic T cells, natural killer cells, CD38cells and interleukin II receptor expression in comparison to a baselinevalue.
 3. A method of inhibiting cortisol-induced suppression ofcytokine production in a subject by: treating said subject with aninitial dosage regimen of enkephalin peptides; discontinuing saidinitial dosage regimen with no further administration of said enkephalinpeptides for at least four weeks; and administering booster dosages ofenkephalin peptides thereafter as necessary to maintain said cytokineproduction.
 4. The method of claim 3, wherein said initial dosageregimen comprises SEQ ID NO: 1, either alone or in conjunction withother therapies, wherein each dose in the initial dosage regimen is 1 to250 μg/Kg bodyweight of the subject, and wherein said initial dosageregimen inhibits cortisol-induced suppression of cytokine production,wherein said at least four weeks of discontinuing said initial dosageregimen with no further administration of said enkephalin peptide is fora period of 4 to 24 weeks, and wherein the inhibition in saidcortisol-induced suppression of cytokine production is maintained duringsaid period in which said administration of said enkephalin peptide isdiscontinued.
 5. A method of inhibiting a cortisol-induced reduction ofgene transcription of IL2 in a subject by: treating said subject with aninitial dosage regimen of enkephalin peptides; discontinuing saidinitial dosage regimen with no further administration of said enkephalinpeptides for at least four weeks; and administering booster dosages ofenkephalin peptides thereafter as necessary to maintain said IL2 genetranscription.
 6. The method of claim 5, wherein said initial dosageregimen comprises SEQ ID NO: 1, either alone or in conjunction withother therapies, wherein each dose in the initial dose regimen is 1 to250 μg/Kg bodyweight of the subject, and wherein said initial dosageregimen inhibits cortisol-induced reduction of gene transcription ofIL2, wherein said at least four weeks of discontinuing said initialdosage regimen with no further administration of said enkephalin peptideis for a period of 4 to 24 weeks, and wherein the inhibition in saidcortisol-induced reduction of gene transcription of IL2 is maintainedduring said period in which said administration of said enkephalinpeptide is discontinued.
 7. A method of inhibiting cortisol-induceddown-regulation of cytotoxic cells or NK lymphocytes in a subject by:treating said subject with an initial dosage regimen of enkephalinpeptides; discontinuing said initial dosage regimen with no furtheradministration of said enkephalin peptides for at least four weeks; andadministering booster dosages of enkephalin peptides thereafter asnecessary to maintain levels of said cytotoxic cells or NK lymphocytes.8. The method of claim 7, wherein said initial dosage regimen comprisesSEQ ID NO: 1, either alone or in conjunction with other therapies,wherein each dose in the initial dosage regimen is 1 to 250 μg/Kgbodyweight of the subject, and wherein said initial dosage regimeninhibits cortisol-induced down-regulation of cytotoxic cells or NKlymphocytes, wherein said at least four weeks of discontinuing saidinitial dosage regimen with no further administration of said enkephalinpeptide is for a period of 4 to 24 weeks, and wherein the inhibition insaid cortisol-induced down-regulation of cytotoxic cells or NKlymphocytes is maintained during said period in which saidadministration of said enkephalin peptide is discontinued.
 9. The methodof claim 2, wherein the dosage of enkephalin peptide during the initialdosage regimen is 20 μg/Kg bodyweight of the patient.
 10. The method ofclaim 1, wherein the need for periodic booster shots is determined bymeasuring the patient's reduction in said cortisol-inducedimmunsuppression from 4 to 16 weeks after cessation of the initialdosage regimen, and comparing the reduction in said cortisol-inducedimmunsuppression with both a baseline value and a response valuemeasured at the end of the dosing schedule.
 11. The method of claim 1,wherein periodic booster dosages are given, as needed, up to 5 times aweek for 1 to 4 weeks.
 12. The method of claim 1, wherein the boosterdosage of enkephalin peptide after the initial dosage regimen rangesfrom 20 to 100 μg/Kg bodyweight.
 13. The method of claim 1, wherein theinitial dosage regimen is 1-5 times per week over a 4-12 week period.14. The method of claim 1, wherein the other therapies include cytokine,antiviral, antibiotics, antifungal, antiparasite and anti-tumortherapies.
 15. The method of claim 1, wherein said initial dosage regimeis once per week for 4 to 12 weeks.
 16. The method of claim 3, whereinthe dosage of enkephalin peptide during the initial dosage regimen is 20μg/Kg bodyweight of the patient.
 17. The method of claim 3, wherein saidinitial dosage regime is once per week for 4 to 12 weeks.
 18. The methodof claim 5, wherein the dosage of enkephalin peptide during the initialdosage regimen is 20 μg/Kg bodyweight of the patient.
 19. The method ofclaim 5, wherein said initial dosage regime is once per week for 4 to 12weeks.
 20. The method of claim 7, wherein the dosage of enkephalinpeptide during the initial dosage regimen is 20 μg/Kg bodyweight of thepatient.
 21. The method of claim 7, wherein said initial dosage regimeis once per week for 4 to 12 weeks.